CN105524936B - The trehalose synthase and its expressing gene of a kind of mutation and application - Google Patents

Abstract

The invention relates to a mutant trehalose synthase, its expression gene and application. The nucleotide sequence of the expression gene of the mutant trehalose synthase is shown in SEQ ID NO.1. The amino acid sequence of the mutant trehalose synthase is shown in SEQ ID NO.2. Based on the three-dimensional structure predicted by (Pseudomonas stutzeri) Qlu3 for the first time, the present invention conducts site-directed mutation of key amino acids in its active center to obtain a mutant protein of trehalose synthase. Large size, high purity, good thermal stability, compared with the wild-type trehalose synthase, the trehalose conversion rate of the mutant is increased by 4.5%, and the production of by-product glucose is reduced by 69.4%.

Description

A mutant trehalose synthase and its expression gene and application

technical field

The invention relates to a mutant trehalose synthase and its expression gene and application, belonging to the technical field of genetic engineering.

Background technique

Trehalose is a very safe and reliable non-reducing disaccharide. Frederick took the lead in exploring its chemical structure by using nuclear magnetic resonance technology. There are two glucopyranose monomers linked by 1,1 glycosidic bonds. of disaccharides. In the first half of the nineteenth century, trehalose was first extracted from a rye ergot fungus by Victrex, and it has been confirmed that trehalose has non-specific protective effects on various biologically active substances. And because of its drought resistance, low temperature resistance, and severe cold resistance, it is called "sugar of life" by many people. Trehalose can enable organisms to endure harsh environmental conditions such as high temperature, high cold, high osmotic pressure, and dehydration due to the fact that it can produce a membrane that attaches to the cell surface to better maintain protein molecules from denaturation, thereby making the animal The characteristics of plant life can exist. This unique functional property makes trehalose not only an excellent active protectant for protein drugs, enzymes, vaccines and other biological products, but also an important ingredient for maintaining cell activity and moisturizing cosmetics. Unique food ingredients for fresh flavor of food and improvement of food quality. Therefore, trehalose can be widely used in medicine, cosmetics and food industries, and has attractive development and application prospects and huge economic benefits.

In view of the wide and important application value of trehalose, the research on finding efficient, convenient and low-cost production methods of trehalose has been widely valued. At present, the production methods of trehalose mainly include yeast extraction method, fermentation method and enzyme synthesis method. Among them, the enzymatic production of trehalose has the characteristics of high specificity, rapidity and mildness, and has become a hot spot in the research and development of trehalose industrial production and one of the feasible ways to achieve short-term results.

Trehalose synthase (TreS) is an intramolecular glucosidic transferase, which can convert the α-1,4 glycosidic bonds of maltose into α-1,1 glycosidic bonds to generate trehalose in only one step. The enzyme reaction process is short, easy to control, does not need to consume high-energy substances, does not require the coexistence of phosphate, and only needs one enzyme to react in one step to obtain trehalose. Therefore, the trehalose synthase conversion method is suitable for industrial production of trehalose. It has a good application prospect and has received extensive attention. So far, more than 15 kinds of microorganisms capable of producing trehalose synthase have been reported at home and abroad. The catalytic efficiency and enzymatic properties of trehalose synthase from different microbial sources are different, but they are all accompanied by the generation of by-products during the reaction process, some as high as 20%, generally about 5%-10%, which is very important The separation and purification of downstream products brings difficulties and increases the production cost of trehalose.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a mutant trehalose synthase and its expression gene and application. The generation of by-product glucose of the mutant trehalose synthase is reduced, and the conversion rate of trehalose is increased. The trehalose synthase is obtained from the trehalose synthase derived from Pseudomonas stutzeri Qlu3 through site-directed mutation A309E.

Technical scheme of the present invention is as follows:

An expression gene of a mutant trehalose synthase, the nucleotide sequence of which is shown in SEQ ID No.1.

A mutant trehalose synthase, the amino acid sequence is shown in SEQ ID NO.2.

The trehalose synthase of the present invention mutates the alanine at position 309 of the key amino acid into glutamic acid by the method of Quick change site-directed mutation, and then transfers it into Escherichia coli for high-efficiency expression. A series of purification methods such as exchange chromatography and molecular sieve can finally obtain high-purity trehalose synthase mutant protein. The above-mentioned recombinant protein can reach the maximum conversion rate after reacting for 2 hours, the conversion rate of trehalose can reach 74.7%, and the by-product is only 1.1%. The trehalose conversion rate of the above-mentioned recombinant protein can still reach 51.2% when placed in a metal bath at 50°C for 30 minutes. The optimum reaction temperature of the above-mentioned recombinant protein is 35°C. The optimum reaction pH of the above-mentioned recombinant protein is 7.0-8.0.

A recombinant vector inserted into the expression gene of the above mutant trehalose synthase.

A transgenic cell line containing the above-mentioned recombinant vector.

Application of the expression gene, recombinant vector or transgenic cell line of the above mutant trehalose synthase in the preparation of trehalose synthase.

Application of the above mutant trehalose synthase in the preparation of trehalose.

The trehalose synthase described in the present invention is obtained from Pseudomonas stutzeri (Pseudomonas stutzeri) Qlu3, and the specific method is: transforming the multi-cloning site of the commercialized pET-15b vector, and deleting redundant enzymes Cutting site, keep the BamHI, XhoI, EcolI, HindIII restriction site, and add the Prescission restriction site to obtain the pGLO1 vector. The gene was connected to the pGLO1 vector to construct a plasmid, and then the alanine (A) at position 309 of the key amino acid was mutated to glutamic acid (E) by the method of Quick change site-directed mutagenesis, and transformed into Escherichia coli BL21DE (3) Perform prokaryotic expression. Then, high-purity mutant trehalose synthase is obtained by separating and obtaining high-purity mutant trehalose synthase through nickel column affinity chromatography, Source-Q ion exchange chromatography and Superdex-200 molecular sieve chromatography.

Beneficial effect

Based on the three-dimensional structure predicted by (Pseudomonas stutzeri) Qlu3 for the first time, the present invention conducts site-directed mutation of key amino acids in its active center to obtain a mutant protein of trehalose synthase. Large, high purity, good thermal stability, and the conversion rate of trehalose increased from 71.5% of the wild type to 74.7%, and the by-products were reduced from 3.6% of the wild type to 1.1%. Compared with the wild type trehalose synthase, the mutant The conversion rate of trehalose increased by 4.5%, and the generation of by-product glucose decreased by 69.4%. The separation cost of trehalose and glucose can be reduced, which lays a foundation for the industrial production of trehalose; and also provides a feasible method for improving the conversion rate of other similar proteins.

Description of drawings

Fig. 1 is the photo of PCR amplification and agarose gel electrophoresis result;

In the figure: M, Marker, T is the target band of TreS-pET-15b;

Figure 2 is a picture of the SDS-PAGE electrophoresis results of trehalose synthase after nickel column purification;

In the figure, ce: whole bacterial solution after crushing, s: supernatant after high-speed centrifugation, elu: protein after nickel column elution;

Fig. 3 is the peak shape result figure of the standard sample of glucose, maltose and trehalose measured by high performance liquid phase;

Fig. 4 is the peak shape result diagram of glucose, trehalose and maltose after the reaction is determined by high performance liquid phase;

Fig. 5A is a curve diagram of the optimal reaction temperature for trehalose synthase enzyme activity after purification;

Fig. 5B is a temperature stabilization curve of trehalose synthase enzyme activity after purification;

Fig. 6A is a graph showing the optimal reaction pH curve of trehalose synthase after purification;

Fig. 6B pH stability curve of trehalose synthase after purification;

Fig. 7A is a graph showing the influence of the reaction time between the purified trehalose synthase and the mutant on the conversion rate of trehalose;

In the figure: TreS is the wild-type trehalose synthase, TreSA309E is the mutant trehalose synthase;

Fig. 7B is a graph showing the influence of the reaction time between the purified trehalose synthase and the mutant on the glucose conversion rate;

In the figure: TreS is the wild-type trehalose synthase, and TreSA309E is the mutant trehalose synthase.

Detailed ways

The technical solutions of the present invention will be further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto.

Example 1: Cloning the mutated trehalose synthase gene.

The primers at the mutation point were designed according to the full-length nucleotide sequence of Pseudomonas stutzeri (Pseudomonas stutzeri) trehalose synthase published on NCBI, and the primer sequences were as follows:

Upstream primer: GTGGCCCTCCGACCAttcGGTGCC

Downstream primer: CGTGCCGAGGGCACCgaaTGGTCG

Using the constructed tres-pGLO1 plasmid as a template, use the above primers for PCR amplification. The PCR reaction system is as follows:

The above-mentioned PCR reaction is carried out according to the following procedures:

Pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 58°C for 30s, extension at 72°C for 4min, 25 cycles; final extension at 72°C for 10min.

After PCR, the fragment length was analyzed by 1wt% agarose gel electrophoresis.

Example 2: Transforming the mutated trehalose synthase gene into an expression host to obtain a positive expression strain.

The PCR product was cleaved by DpnI endonuclease, and the enzyme cleavage reaction system was as follows:

Enzyme digestion system for PCR products:

Mix well and centrifuge for a few seconds, collect the droplet on the tube wall to the bottom of the tube, and react at 37°C for 30 minutes.

Transformation of recombinant plasmids

(1) Preparation of Competent Cells

①Pick a single colony of Escherichia coli BL21 (DE3) (purchased from Shanghai Qianchen Biotechnology Co., Ltd.) (or pick a preserved strain) and inoculate it into 10ml of liquid LB medium, culture overnight at 37°C and 210rpm;

② Inoculate 5ml of bacterial liquid into 500ml of LB medium, shake at 37°C, 210rpm for 70-80min until OD ₆₀₀ reaches 0.375;

③ Place the bacterial solution on the ice-water mixture for 10 minutes, and pre-cool the 50ml centrifuge tube at the same time;

④ Transfer the bacterial solution to a centrifuge tube, collect the bacterial cells at 4°C, 3700 rpm, and 10 minutes, and discard the supernatant;

⑤ Add about 10ml of ice-cold activation buffer (0.1M CaCl ₂ ) to each centrifuge tube, break up the precipitate with a sterilized 5ml tip, and then add about 30ml of ice-cold activation buffer to each tube. Buffer, mix by inverting, let stand on ice for 20min;

⑥Centrifuge at 4°C, 3700rpm for 10min; discard the supernatant, pour out the residual liquid, and break up the precipitate according to the amount of 500ml bacterial solution and 12ml ice-precooled storage buffer (0.1M CaCl ₂ , 15% glycerol by volume). Transfer once, then blow to break up).

⑦ Aliquot the competent cells into ice-precooled sterilized EP, 100 microliters per tube, and place on ice (0°C) for 30 minutes.

⑧ Competent cells were frozen at -80°C to obtain competent cells BL21(DE3).

Note: Try to keep the cells at low temperature during the whole process. The gun tip, centrifuge tube, EP tube and buffer used must be sterilized. The whole process is operated in an ultra-clean bench. After the competent cells are finished, test their efficiency and whether they are Bacteria.

(2) Conversion of ligated products

① Add 15 μL of the ligation product to 100 μL of freshly prepared competent cells BL21DE(3), mix gently, and place on ice for 30 minutes.

②Heat shock at 42°C for 90s, then quickly place in an ice bath to cool for 3min.

③ Add 200 μL LB liquid medium, shake and culture at 37°C, 180 rpm/min for 60 min, so that the bacteria can return to normal growth state and express the antibiotic resistance gene encoded by the plasmid;

④Take 200 μL of the above bacterial solution and spread it on the resistant LB solid medium (ampicillin 100 mg/L).

⑤ After the bacterial solution is blotted dry, invert the plate and incubate at 37°C for 12-16 hours.

The components of LB liquid medium are as follows:

5g yeast powder, 10g peptone, 10g NaCl, add water to 1L, and autoclave at 121°C for 20min for later use.

The components of LB solid medium are as follows:

5g yeast powder, 10g peptone, 10g NaCl, 2% agar powder, add water to 1L, and autoclave at 121°C for 20min for later use.

Identification of positive clones

(1) Colony PCR identification

Pick a single colony, shake and culture at 37°C for 6-8 hours, absorb 1 μL of the bacterial liquid, and carry out PCR identification according to the 15 μL PCR reaction system. If it is a positive clone, a band can be detected by agarose gel electrophoresis, as shown in Figure 1.

(2) Protein expression and solubility identification

Add IPTG (isopropylthiogalactopyranoside) at a final concentration of 0.2mM to the remaining bacterial solution identified by colony PCR, induce expression for 1 hour, centrifuge at 12,000 rpm/min for 1 minute, discard the supernatant, and collect the bacteria. Add 2 times of loading buffer, suspend the pellet with a gun tip, and denature at 95°C for 10 minutes. If it is a positive clone, protein overexpression can be detected by SDS-PAGE.

(3) DNA sequencing

The positive clones identified by the above two methods were sequenced correctly, and the nucleotide sequence inserted in the positive clones obtained is shown in SEQ ID NO.1.

Example 3: Fermentation and culture of positive expression strains, isolation and purification of mutated trehalose synthase recombinant protein

Seed culture: Pick the positive clones prepared in Example 2 in a conventional way, place them in 5 mL of LB liquid medium containing 100 μg/L ampicillin, and culture them with shaking at 37°C for 5-6 hours;

Bacteria expansion culture: Inoculate the seeds into 1L of liquid medium containing 100mg of ampicillin, culture with shaking at 37°C until the bacterial concentration OD ₆₀₀ is 1.0, then cool down to 16°C; add isopropanol with a final concentration of 0.2mM after 1 hour thiogalactoside (IPTG), induced overnight.

Collect bacteria: 4200rpm, centrifuge at 4°C for 15min, discard the supernatant, and harvest the bacteria; add resuspension solution (25mM Tris-HCl, pH8.0, 200mM NaCl), shake and precipitate the bacteria cells; add protease inhibitor PMSF (Phenylmethylsulfonyl fluoride, benzoic acid sulfonyl fluoride) to a final concentration of 2 mM.

Ultrasonic disruption of bacterial cells: ultrasonic 3s, interval 6s, 500W, work 60 times.

Ultracentrifugation: after sonication, the cell disruption solution was centrifuged at 14000rpm, 4°C for 45min, and the supernatant was collected for the next step of separation and purification.

Ni-NTA affinity chromatography: Pour the collected supernatant containing soluble protein into the regenerated nickel column; after the supernatant flows out, wash buffer (25mM Tris-HCl, pH8. imidazole) to wash 10 column volumes to remove non-specifically adsorbed proteins; finally use elution buffer (25mM Tris-HCl, pH8.0, 100mMNaCl, 250mM imidazole) to elute the target protein and collect it in a clean pre-cooled beaker; use SDS -PAGE electrophoresis to detect whether the protein is soluble, whether the soluble protein can be combined with Ni-NTA, whether it can be eluted, and the concentration of the protein. The results are shown in Figure 2.

Anion-exchange chromatography purification (Source-Q): Dilute the soluble protein removed by Ni-NTA affinity chromatography with solution A (25mM Tris-HCl, pH8.0) for 3 to 4 times, and then load the sample on the Solution A was equilibrated on the ion exchange column SourceQ, using solution A and solution B (25mM Tris-HCl, pH 8.0, 1M NaCl) for linear gradient elution. Observe the change of the absorbance value (A280) at 280nm, collect the collection tubes near each peak position, and perform SDS-PAGE electrophoresis in order to obtain the target protein.

Molecular sieve purification: Judging the properties of the protein on the ion exchange column according to the shape of the protein peak on the ion exchange column, whether there is a "shoulder" and whether it is symmetrical and sharp. Concentrate the protein with good properties to 2mL by ultrafiltration, and load it on the gel filtration chromatography column Superdex-200 that has been equilibrated with solution C (25mM Tris-HCl, pH8.0, 100mM NaCl), with a flow rate of 0.4mL /min. The protein peaks were collected and subjected to SDS-PAGE electrophoresis to detect the purity and properties of the protein. After sequencing, the amino acid sequence is shown as SEQ ID NO.2, which is the mutant trehalose synthase.

Example 4: Enzyme activity assay method of mutated trehalose synthase

150 mM maltose, 20 mM Na2HPO ₄ -NaH ₂ PO ₄ buffer solution pH 7.0, 1 μM of the mutated trehalose prepared in Example 3 were added to the reaction system, and reacted at 37° C. for 2 h. The conversion rate of trehalose was determined by the method of high-pressure liquid phase, and an amino column was used in the determination process; the column temperature was 40°C, and the mobile phase was a mixed solution of acetonitrile and water, and the volume ratio of the two was 3:1; the flow rate was 1mL/min; The detector is a differential detector; the detection time is 25min. Standard test results are shown in Figure 3 and the conversion rate is calculated according to the following formula:

According to the high performance liquid phase results shown in Figure 4, the maltose peak area, trehalose peak area and glucose peak area are fitted to the curve by software, and the mass of the three is calculated, wherein m3 is the mass converted into trehalose, and m2 is the mass converted into The quality of glucose, m1 is the quality of remaining maltose.

Example 5: Determination of Biochemical Properties of Mutant Trehalose Synthase

Determination of the optimum reaction temperature:

After diluting the enzyme solution to an appropriate multiple, react at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C for 2 hours, and use the method of high pressure liquid phase to determine the concentration of trehalose Enzyme conversion rate. The results are shown in Figure 5A. The optimal reaction temperature of trehalose synthase recombinant protein is 35°C.

Determination of temperature stability:

After diluting the enzyme solution by a certain number of times, incubate at 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 45°C, 50°C, 55°C, 60°C for 30 minutes, add the substrate maltose, and then react 2h, measure the conversion rate of trehalose. The results are shown in Figure 5B. Trehalose synthase still maintains 70% of the enzyme activity when it is placed in a metal bath at 50°C for 30 minutes.

Determination of the optimum reaction pH value:

Use pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 0.10.5, 11. A series of phosphate buffer dilutions of recombinant trehalose synthase mutants. Then put it at 35°C for 2h to measure the enzyme activity. The results are shown in Figure 6A.

Determination of pH stability:

After diluting the enzyme solution by a certain number of times, dilute the recombinant trehalose with a series of phosphate buffers of pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 0.10.5, 11 Synthase mutants. Incubate at 35°C for 30 minutes, add the substrate maltose, then react for 2 hours, and measure the conversion rate of trehalose. The results are shown in Figure 6B. The trehalose synthase mutant maintains more than 95% of its enzyme activity during the pH7.0-8.0 period.

Example 6: Determination of optimal reaction time of mutated trehalose synthase

The reaction system is 150mM maltose, 20mM Na ₂ HPO ₄ -NaH ₂ PO ₄ pH7.2, 1μM trehalose synthase mutant recombinant protein, react at 37°C, at 5min, 20min, 40min, 1h, 2h, 3h, 4h Samples were taken at different times, and then detected by HPLC. The detection method was the same as in Example 4. The conversion rate is obtained by calculation. The conversion rate of trehalose can reach 74.7% and the conversion rate of glucose is 1.1% after the reaction of the mutant trehalose synthase. As shown in Figure 7.

Comparative Experiment

Since the trehalose synthase mutant improves the conversion rate of the trehalose synthase, the by-products are reduced. Dilute the trehalose synthase enzyme solution before and after the mutation to an appropriate multiple, react at 37°C, take samples at different times of 5min, 20min, 40min, 1h, 2h, 3h, and 4h, and then use HPLC for detection. The detection method is the same as the implementation Example 4. The conversion rate is obtained by calculation. The preparation method of the trehalose synthase protein is simple, the yield is large, and the purity is high; the experiment verifies that it has good thermal stability, and the conversion rate of trehalose is increased from 71.5% of the wild type to 74.7%, the result is shown in Figure 7A; and by-products The conversion rate of glucose from 3.6% in wild type decreased to 1.1%, as shown in Fig. 7B. Compared with trehalose synthase before mutation, the trehalose conversion rate of the mutant increased by 4.5%, while the production of by-product glucose decreased by 69.4%. The separation cost of trehalose and glucose can be reduced, which lays the foundation for the industrial production of trehalose; and also provides a feasible method for improving the conversion rate of other similar proteins.

Claims (7)

1. a kind of expressing gene of the trehalose synthase of mutation, nucleotide sequence is as shown in SEQ ID NO.1.

2. a kind of trehalose synthase of mutation, amino acid sequence is as shown in SEQ ID NO.2.

3. a kind of recombinant vector, expressing gene described in claim 1 is inserted in expression vector.

4. recombinant vector as claimed in claim 3, which is characterized in that the expression vector is pET-15b.

5. a kind of transgenic cell line contains the recombinant vector described in claim 3.

6. the recombinant vector or right described in the expressing gene for the trehalose synthase being mutated described in claim 1, claim 3 are wanted The transgenic cell described in 5 is asked to tie up to the application prepared in trehalose synthase.

7. application of the trehalose synthase of the mutation described in claim 2 in preparing trehalose.